1. Running the BioLogic DuoFlow Starter Kit
Bio-Rad Life Science Research Group
Product and Application Tutorials
Thank you for participating in this webinar on running the BioLogic DuoFlow Starter Kit! This webinar is presented to you by the Technical support Team of Bio-Rad laboratories.

2. For direct assistance, call Technical Support at:
Welcome!
Welcome to the Bio-Rad Laboratories Biologic DuoFlow Starter Kit webinar.
The DuoFlow Chromatography System offers flexibility with multiple system configurations and optional upgrades, all using a common software platform that is user friendly and easy to follow.
This webinar will teach you the basics of running the standard DuoFlow Chromatography System by following the instructions for the DuoFlow Starter Kit (catalog # ) on your system with the UNO Q1 Column that came with it.
For direct assistance, call Technical Support at:
, Option #2
The DuoFlow Chromatography System offers flexibility with multiple system configurations and optional upgrades, all using a common software platform that is user friendly and easy to follow.
This webinar will teach you the basics of running the standard DuoFlow Chromatography System by following the instructions for the DuoFlow Starter Kit (catalog # ) on your system with the UNO Q1 Column that came with it.
For direct assistance, call Technical Support at:
, Option #2

3. The DuoFlow Desktop Icons
To Start, you need to configure your system. Select the BioLogic Configuration icon first.

4. Configuring DuoFlow With or Without Maximizer
Select the specifications and components of your system.
Here we are with no Maximizer installed, choosing the F10 pump, and the Model 2128 Fraction Collector.

5. Start BioLogic DuoFlow Software
Now you are ready to launch the BioLogic DuoFlow software by clicking on this icon.
You will then come to the Manual Screen, which is the first screen displayed when the software is started.

6. Manual Screen
The Manual Screen.
This screen is grouped into the system menus comprising the drop-down menus along the top and the toolbar just below it, the large control window in the center, and the status bar along the bottom.
The control window contains the control panels for the instruments connected to the DuoFlow in the top section, followed by the valve control for both the workstation and the Maximizer, if one is installed, with the chromatogram display control just below that.
As seen on this screen, the QuadTec UV detector is selected, and the circled arrow can switch between it and the basic optics module if your system has both.

7. The Manual Screen – The Resize Button
To enlarge the chromatogram view screen on the Manual screen display, select the Resize button to the right of the chromatography display.
To set both of the Y-axis, use the scroll bars by the sides of the chromatogram display.

8. Manual Screen
This next screen shot view shows the optics module selected for UV detection.
Note that many menu and tool bar functions are grayed out and inaccessible until you select or enter a user name in the browser. Select Browser from the toolbar and this will list the users.

9. Browser Screen
Here we see 2 listed users.

10. Browser Screen
Clicking the + will show you the User Icons of the named users, which are Demo Chromatography and Default in this case.

11. Browser Screen
As we click the + by Demo Chromatography, the Project Icons show the projects for this user. This file hierarchy extends down to the individual methods and runs for that method.

12. Browser Screen
Under Multi-Dimensional Chromatography Demo Files, we see Affinity-Desalting and Affinity (5 x 1 ml) –Desalting-Ion Exchange.

13. Browser Screen
Under the Affinity-Desalting method icon ( a Queue Icon in this case), we see the Run Icons.

14. Browser Screen - File Hierarchy
Here is an example of a complete tree from User all the way down to the individual Runs.

15. Create a New Method Select the Browser icon from the tool bar menu.
For our demonstration, in the Manual screen, select the Browser Icon from the tool bar. In the Browser screen you will enter a user name for your method (refer to page 6-1 of the DuoFlow Instruction Manual for more information on the Browser screen) according to the following steps:
Select the Browser icon from the tool bar menu.

16. Create a New Method Select the New icon from the upper left
side of the Browser screen.
Select New from the drop-down menu
and enter your name in the dialog box.
Select the New icon from the upper left side of the Browser screen.
Select New from the drop-down menu and enter your name in the dialog box.

17. Create a New Method Click on the Project icon for your user name.
Select New and New Method. Enter
your method name (or use default
Method 1.
Click OK to proceed to the
instrument/devices Setup screen.
Click on the Project icon for your user name.
Select New and New Method. Enter your method name (or use default Method 1).
Click OK to proceed to the instrument/devices Setup screen.

18. Program the Instrument Set-Up
In the Setup screen select the instrument and devices to be used for the Starter Kit method. The icons grouped on the left side of the screen (refer to Figure 3, Available Devices) show all the instruments and devices that can be connected to the BioLogic DuoFlow systems.
The list of devices in the right box (Devices in Setup) identifies those devices selected for use with a specific method. The initial Devices in Setup are a UV detector, conductivity monitor, and an AVR7-3 valve. These come standard with the BioLogic DuoFlow system. The DuoFlow QuadTec system includes a QuadTec UV/Vis detector in place of a UV detector.

19. Program the Instrument Set-Up - Selecting Fraction Collector
Click on the Fraction Collector button in the Available Devices box. A dialog box will appear asking you to choose the type of collector; i.e., a generic collector, a model 2110, or a BioFrac. Click on BioFrac and click the OK button. You will now see BioFrac fraction collector in the Devices in the Setup box. The F1 Rack (12-13 mm tubes) is automatically selected.

20. Program the Instrument Set-Up - Selecting Detector
If you are using a QuadTec UV/Vis detector, click on the Detectors button in the Available Devices box.
A dialog box will appear asking you to choose a detector. In this case we will select the QuadTec and check each of the four wavelength boxes.
Enter the wavelengths: (1) 280 nm, (2) 260 nm, (3) 214 nm, and
(4) 405 nm. Press OK.

21. Program the Instrument Set-Up - Labeling the Buffers
In the Buffer A field, type in 25 mM Tris-HCl, pH 8.1.
In the buffer B field, type in 25 mM Tris-HCl M NaCl, pH 8.1.
Prepare the anion exchange standard and the buffers A and B from the Starter Kit as described in Section 1 of the Starter Kit Instruction Manual. Use degassed buffers if at all possible for best results.
In the Gradient Pump section of the setup screen, enter you buffer names.
In the Buffer A field, type in 25 mM Tris-HCl, pH 8.1.
In the buffer B field, type in 25 mM Tris-HCl M NaCl, pH 8.1.

22. Program the Instrument Set-Up - Saving the Method
Save the device setup
The Setup is now complete.
To save the device setup, choose Save Setup under the File menu and enter a name for your setup.
Immerse the workstation pump A and B inlet lines in a container of HPLC grade water. Prime both pumps as described in the Starter Kit Manual. Move the AVR7-3 Inject Valve to the purge position, and purge both pumps for 2 minutes.

23. Program the Instrument Set-Up – Program the Method
Protocol
Put the AVR7-3 valve to the load position and flush the system. When buffers A and B are prepared, install the UNO Q1 Column.
Repeat prime and purge protocol with the pump inlets immersed in the correct buffers with the AVR7-3 in the purge position. When done, switch back to the load position and equilibrate column with 5 CVs of buffer B, followed by 10 CVs of buffer A at 2 ml/min.
You are now ready to program the separation steps for your method. Click on the Protocol button.

24. Program the Instrument Set-Up – Program the Method
Protocol
Program the separation
method shown in Figure 4
of the Starter Kit Manual.
Set the UV range to 0.1 AUFS and the conductivity range to 100 mS/cm.
When the column equilibration is done, the conductivity monitor on the status bar should read at about 3 mS/cm or less.
Program the separation method shown in Figure 4 of the Starter Kit Manual.

25. Program the Instrument Set-Up – Program the Method
Protocol
From the left side of the screen, press the fraction collection icon.

26. Program the Instrument Set-Up – Program the Method
Protocol
In the pop-up window that appears, choose Collect All with a fraction size of 2.00 ml and a delay of Note that values can be entered with the cursor or by clicking the up and down arrows by the window. If the increments of the arrows are too low or high, enter the value with the cursor. This will be Step 1.
Make sure the correct rack type is displayed.

27. Program the Instrument Set-Up – Program the Method
Protocol
Program the remaining steps using the Add Step icons from the left side of the screen.
Program the remaining steps using the Add Step icons from the left side of the screen.

28. Program the Instrument Set-Up – Program the Method
Protocol
Step Number Start (ml) Step
Collect fractions of size 2.00 ml during the entire run
As each step is added, you will see it added with a step number, a volume amount from where that step starts in context of the entire run, and a description of that step.
Click the Fraction Collector button and set it to collect fractions of 2.0 ml size during the entire run.

29. Program the Instrument Set-Up – Program the Method
Protocol
Step Number Start (ml) Step
Isocratic flow with 100% 25 mM Tris-HCl, pH 8.1,
0% 25 mM Tris-HCl, 0.5 M NaCl, pH 8.1 at 4.00 ml/min for 1.0 ml
For Step 2, press the Isocratic Flow button on that same left side of the screen. Select a flow rate of 4.0 ml/min, a volume of 1 ml, and 100% A: 25 mM Tris-HCl, pH 8.1 and 0% B: 25 mM Tris-HCl M NaCl, pH 8.1.

30. Program the Instrument Set-Up – Program the Method
Protocol
Step Number Start (ml) Step
Zero Baseline to set UV baseline to Select either
UV detector or QuadTec detector.
For the third step, press the Zero Baseline button on the left, and Select either the UV detector or QuadTec detector. The accumulated volume of the run so far should read 1.00 ml.

31. Program the Instrument Set-Up – Program the Method
Protocol
Step Number Start (ml) Step
Load inject sample, static loop: Inject 0.5 ml sample at
4.00 ml/min. You will be injecting the loop size of 50 ul.
Now for step 4, press the Load Inject Sample button and select the Static Loop (the 50 ul supplied in the Starter Kit), set the volume to 0.5 ml and keep flow rate at 4.00 ml/min. This will inject 0.5 ml of your A buffer through the 50 ul loop, loading your sample.

32. Program the Instrument Set-Up – Program the Method
Protocol
Step Number Start (ml) Step
Isocratic flow with 100% 25 mM Tris-HCl, pH 8.1, 0% 25 mM
Tris-HCl, 0.5 M NaCl, pH 8.1 at 4.00 ml/min for 0.8 ml
The fifth step will add an isocratic buffer A column wash with 100% buffer A for 0.8 mls at 4.00 ml/min. Press the Isocratic Flow button and select 100% Buffer A at 4.00 ml/min for 0.8 mls. The accumulated volume of the run should now read 1.50 ml.

33. Program the Instrument Set-Up – Program the Method
Protocol
Step Number Start (ml) Step
Linear gradient with 0% to 50% 25 mM Tris-HCl,
0.5 M NaCl, pH 8.1 at 4.00 ml/min for 13.0 ml
Step 6 begins the gradient with pump B. Select the Linear Gradient button and make the initial percentage of Buffer A 100% and the final as 50% and the volume to ml. Make the initial percentage of Buffer B as 0% and the final as 50% also with a ml volume. The accumulated volume of the run should now read 2.30 ml.

34. Program the Instrument Set-Up – Program the Method
Protocol
Step Number Start (ml) Step
Isocratic flow with 0% 25 mM Tris-HCl, pH 8.1, 100%
25 mM Tris, 0.5 M NaCl, pH 8.1 at 4.00 ml/min for 2.8 ml
Step 7 adds a 100% B step to elute the final protein from the Anion Exchange Standard. Press the Isocratic Flow button and select 100% Buffer B at 4.00 ml/min for 2.8 mls. The accumulated volume of the run should now read ml.

35. Program the Instrument Set-Up – Program the Method
Protocol
Step Number Start (ml) Step
Isocratic flow with 100% 25 mM Tris-HCl, pH 8.1, 0%
25 mM Tris, 0.5 M NaCl, pH 8.1 at 4.00 ml/min for 8.0 ml
End of protocol
Step 8 will re-equilibrate the column in Buffer A. Press the Isocratic Flow button and select 100% Buffer A at 4.00 ml/min for 8.00 mls. The accumulated volume of the run will now read ml. After this the protocol will end with a final total volume of 26.1 ml.

36. Program the Instrument Set-Up – Program the Method
Protocol
The final written method should look like this

37. Program the Instrument Set-Up – Program the Method
Protocol
When you have finished programming the method, press the toolbar button RUN.
A dialog box will ask you to name the run. Accept the default Run 1 and click
the OK button. You will now see the Run Screen.

38. Program the Instrument Set-Up – Program the Method
Protocol
Start your method by pushing start button with the green triangle along the top of the Chromatogram display.

39. Program the Instrument Set-Up – The Run Screen
The toolbar buttons on the left side of the screen enable you to check that the screen display ranges for UV (Optics Module), QuadTec UV/Vis and conductivity are correctly set and that the gradient pump pressure limits are appropriate (700 psi high limit and 20 psi low limit) for the UNO Q1 column.

40. The Run Screen – Status Bar and Zeroing the Baseline
If you have been equilibrating the column while writing the method, you will see that the Status Bar is displaying the flow rate and UV values, QuadTec UV/Vis, and conductivity detectors (see red arrow). To zero the UV or QuadTec, click on the Zero Baseline button in the appropriate box. It may be selected at any time.

41. The Run Screen – Changing the Scale
To scale the on-screen chromatogram trace display, use the scroll bars located on the left and right axis of the chromatogram window.

42. Chromatograms from the Basic Starter Kit
Results for both the standard UV and QuadTec should look like these.
Congratulations! You have completed the running of the DuoFlow Starter Kit!
Standard UV QuadTec

43. Part Numbers Catalog # Description 760-0135 Starter Kit
BioLogic DuoFlow Standard System
BioLogic Maximizer Kit
BioLogic QuadTec Kit
BioFrac Fraction Collector
ml Capless Micro Test Tubes, polypropylene, 500/box
x 100 mm Clear Polystyrene Tubes, 1,000/box
x 100 mm Clear Polystyrene Tubes, 1,000/box
UNO Q1 Column, 1.3 ml
Protein Standard for Anion Exchange Chromatography
Here are a list of common parts for the DuoFlow system.

44. Software Help Feature
The software help feature is a very good tool to use in directing you in method creation and running the system. You can display the help menu by pressing the F1 key or using the drop-down menus above the toolbar.